A novel medium for long-term primary culture of hemocytes of Metapenaeus ensis

The development of a suitable shrimp cell medium is essential for achieving a long-term cell culture and finite cell line of shrimps routinely. In this study, we have successfully developed an optimal shrimp cell medium that can be used for long-term in vitro culture and continuous subculture of the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. In this article, we have focused on the details for the preparation of the optimal shrimp cell medium by diluting and mixing of various stock solutions as well as the methods for isolation and primary culture of MeH cells.• A novel shrimp cell growth medium is developed for long-term shrimp hemocytes culture.• The preparation method of shrimp cell growth medium is successfully established.• Obvious cell activity and proliferation potential of isolated shrimp cells can be maintained beyond 30 days.


Table 1
The formulas of the optimal shrimp cell growth medium (SGM) [1] .Note: The medium listed in the table were adjusted to a osmolarity of 620 ± 20 mOsm •kg − 1 and pH value of 7.2 ± 0.2.
l -15 medium components, the SGM medium was optimized by removing all the components of amino acids from l -15 medium and then sequentially adding varied folds of the shrimp serum-based amino acid mixtures, or shrimp serum-based saccharide mixtures, or shrimp serum-based fatty acid mixture and other organic and inorganic supplements such as FBS, gelatin, and so on, which listed in Table 1 .A total of 16 kinds of shrimp cell basic media (SBM) and 16 kinds of SGMs had been prepared and tested to obtain the formulation of the optimal SGM.The detailed optimization process and evaluation of these media can be found in related paper titled by routine development of long-term primary cell culture and finite cell line from the hemolymph of greasyback shrimp ( Metapenaeus ensis ) and virus susceptibility [1] .In the present study only the detailed protocol for the preparation of the optimal SGM is addressed.Importantly, the components of the optimal shrimp cell growth medium (SGM) included 26 kinds of amino acids, 12 kinds of saccharides, 8 kinds of vitamins, 8 kinds of inorganic salts, phenol red, 8.6% (m/v) gelatin, 20% (v/v) shrimp ovary extracts, 4% (v/v) shrimp eye stalk extracts, 15% (v/v) fetal bovine serum (FBS), 20 μg L − 1 epidermal growth factor (EGF), 20 μg L − 1 basic fibroblast growth factor (bFGF), 1.0 × 10 5 IU L − 1 penicillin G sodium, 1.0 × 10 5 IU L − 1 streptomycin and 0.25 mg L − 1 amphotericin B. The formulas of shrimp cell growth medium are listed in details in Table 1 .The formulas of 12 kinds of stock solutions (A-L) used to prepare the optimal SGM is listed in Table 2 .
This optimal SGM is prepared by diluting and mixing various stock solutions and individual nutritional ingredients as follows: (1) To prepare the 12 kinds of stock solutions (A-L), respectively.
(2) To prepare the ovary extract and eye stalk extract of shrimps, respectively.
(3) To dilute and mix the various stock solutions and individual ingredients one by one.(4) To isolate and culture the shrimp hemocytes using the optimal SGM.

Preparation of the stock solutions A-L
For amino acids, as shown in Table 2 , there are 5 kinds of stock solutions (A, B, C, D and E) with a concentration of 100 × needed to prepare, based on the solubility of amino acids (water-, acid-and alkali-soluble) and their contents in the medium.Of them, stock solution A consists of 12 kinds of water-soluble amino acids of glycine, L -serine, l -valine, l -lysine, l -alanine, -alanine, ornithine, l -arginine, l -threonine, hydroxyproline, 4-hydroxybutanoic acid ( -ABA) and l -glutamine.Stock solution B and C are water-soluble taurine and l -proline, respectively.Stock solution D includes 5 kinds of alkali-soluble amino acids of l -histidine, lisoleucine, l -asparagine, l -methionine and l -phenylalanine, which are pre-dissolved in 1 M NaOH and then diluted in water.Stock solution E includes 7 kinds of acid-soluble amino acids of l -cystine, l -leucine, l -tyrosine, l -aspartic acid, l -glutamic acid, l -cysteine and l -tryptophan, which are pre-dissolved in 1 M HCl and then diluted in water.
For saccharides, there are 2 kinds of stock solutions F and G with a concentration of 100 × needed to prepare.Stock solution F consists of 11 kinds of water-soluble saccharides (xylose, fucose, ribose, sucrose, lactose, fructose, arabinose, trehalose, gentiobiose, D + Galactose and sodium pyruvate).Stock solution G contains only glucose which is water-soluble in water.
For vitamins, there are 2 kinds of stock solutions (H and I) with a concentration of 100 × needed to prepare.Stock solution H is made up of 7 kinds of water-soluble vitamins including i-inositol, niacinamide, choline chloride, d -calcium pantothenate, thiamine monophosphate, pyridoxine hydrochloride and riboflavin 5 ′ -phosphate Na.Stock solution I contains only folic acid which is alkalisoluble in 1 M NaOH.
For inorganic salts, there are also 2 kinds of stock solutions (J and K) needed to prepare.Stock solution J is a mixture of 3 kinds of water-soluble inorganic salts of MgSO 4 , KH 2 PO 4 , and Na 2 HPO 4 with a concentration of 100 ×.Stock solution K is a mixture of 4 kinds of water-soluble inorganic salts of NaCl, KCl, MgCl 2 and CaCl 2 with a concentration of 25 ×.Instead of a stock solution, the pH regulator salt of NaHCO 3 is added in powder separately just before used.
The stock solution L which contains phenol red only is prepared as a pH indicator to a concentration of 100 ×.
All the above-mentioned stock solutions are stored at − 80 • C until used.

Preparation of shrimp ovary and eye stalk extracts
As shown in Fig. 1 , the shrimp ovary or eye stalk tissues are aseptically dissected and then homogenized in an ice bath after suspended in sterile water at a ratio of 20 mL per gram tissues, respectively, and then incubated in a 60 • C water bath for 1 h with intermittent oscillation.The homogenates are frozen and thawed once, and then centrifuged at 10,000 × g for 2 h at 4 °C.After that, the supernatant is collected, filter-sterilized (0.22 μm) by a vacuum filtration system (Corning, USA) and stored at − 80 • C until used [2] .

Preparation of the optimal SGM
As shown in Fig. 2 , the gelatin is a water-insoluble powder at room temperature, but it can dissolve and form an aqueous solution when heated to 60 °C, and then, the aqueous solution of gelatin will gel once it cools [3] .Thus, the aqueous solution of gelatin is the first component to be prepared in case of the possible inactivation or degradation of other nutrients by heat.To prepare a 100 mL medium, forty-six mL of gelatin water solution containing 8.6 g gelatin in powder is first prepared and then cools to 37 • C.After that, the 12 kinds of stock solutions (A-L) as listed in Table 2 are diluted and added into the gelatin water solution successively as required and mixed well immediately, followed by the addition of 100 mg NaHCO 3 .After well mixing, the pH value of the medium was adjusted to 7.2 ± 0.2 by 1 M HCl and 1 M NaOH, and the osmotic pressure was adjusted to 620 ± 20 mOsm•kg − 1 by NaCl, and the volume was made up to 100 mL using sterile water.Next, the medium is filter-sterilized (0.22 μm), and then growth factors (EGF and bFGF), antibiotic mixture (penicillin, streptomycin and amphotericin B) are added into the medium as required just before use.The freshly prepared SGM is stored at 4 • C and should be used up within one month.The volume percentage of the stock solutions (A-L) and other supplements are summarized in Fig. 3 and you can use it as a reference in the preparation of SGM.

Isolation and culture of shrimp hemocytes using the optimal SGM
As shown in Fig. 4 , after the shrimp is anesthetized by immersing in 75% ethanol for about 10 s, the surface of the sampling area is successively disinfected with 75% alcohol and iodophor solutions [4] .After drying the surface of the sampling area with sterile paper, shrimp hemocytes are aseptically drawn out with a 2.5-mL syringe preloaded with 0.5 mL anticoagulant solution as listed in Table 3 .Next, the shrimp hemocytes are pelleted by centrifugation at 1000 × g for 10 min and resuspended in 2 × PBS ( Table 3 ) containing 30 × antibiotic mixtures and treated for 10 min.Then after centrifugation, the cell pellet is pooled and washed once with 2 × PBS by centrifugation.Finally, the cell pellet is resuspended with the optimal SGM and seeded into culture plates and incubated at 28 • C with 3% CO 2 , and the medium was half-replaced every 2 or 3 days.Noteworthy, before use, the SGM which is stored at 4 °C refrigerator should be warmed in a water bath to 37 • C to melt it from gelatinous to liquid.It will take ∼15 min for 100 mL SGM to completely melt.Note: The solutions listed in the table were adjusted for osmotic pressure of 620 ± 20 mOsm•kg − 1 and pH value of 7.2 ± 0.2.
Fig. 5.The light micrographs of shrimp hemocytes cultured in the optimal shrimp cell growth medium for 36 (A) and 90 days (B), respectively.Scale bar, 50 μm.

Method validation (1) Formation of confluent and long-term MeH cell monolayer
As shown in Fig. 5 , using the optimal SGM, confluent MeH cell monolayer can be obtained routinely and healthily maintained for a long time, at least 90 days.The MeH cell type was identified by Wright-Giemsa staining, and data for the cell characterization and cell percentage before and after a long-term culture can be found in the related research article [1] .In brief, SGM can support the long-term survival and growth of three types of shrimp hemocytes, namely granulocytes, semi-granulocytes and prohaemocytes, except for the hyalinocytes which possibly had died or differented into granulocytes or semi-granulocytes in the in vitro culture conditions.
Moreover, obvious cytopathic effect (CPE) and replication of shrimp covert mortality nodavirus (CMNV) had been observed in the infected MeH cells cultured in SGM in the test of virus susceptibility, confirming the maintenance of normal cellular functions of shrimp hemocytes in SGM [1] .
More information and culture results can be found in the related research article [1] . (

2) Viability analysis of MeH cells by Calcein-AM staining
As shown in the related research article, the optimal SGM can well support the growth and survival of MeH cells.It was found that by Calcein-AM staining the survival rate of shrimp hemocytes cultured in the optimal SGM for 30 days was 46.24 ± 2.11% [1] , much better than previously published results.Calcein-AM (calcein acetoxymethyl ester) can easily penetrate the living cell membrane because of the addition of acetoxymethyl ester (AM) group to Calcein, which enhances its hydrophobicity.Calcein-AM itself can't emit any fluorescence.After entering the cell, it is hydrolyzed by endogenous esterase in the cell to produce a polar molecule of Calcein, which can emit green fluorescence but can't penetrate the cell membrane and be trapped in the cell.In contrast, dead cells can't hydrolyze Calcein-AM into Calcein, thus can't emit fluorescence because of the lack of active esterase in the dead cells [ 5 , 6 ].In this study, Calcein-AM (Beyotime, Shanghai, China) was used to detect the cellular activity of shrimp hemocytes.Just before staining, the stock solution of Calcein-AM was diluted by serum-free medium into a working concentration of 1 μM.And then the old medium in each well was removed and the cells were washed with 2 × PBS for 1-2 times.Then an appropriate volume of Calcein-AM working solution was added, followed by gently shaking in order to make the dye cover all cells, and then incubated for 45 min at 28 • C in darkness.Generally, the added volume of each well of a 96-well plate is 100 μL, that of 24-well plate is 250 μL, that of 12-well plate is 500 μL, and that of 6-well plate is 1 mL, respectively.After incubation, the cells were washed with 2 × PBS once, re-fed with fresh medium and observed under an inverted fluorescence microscope. (

3) Proliferation potential analysis by EdU staining
As shown in the related research article, the use of the optimal SGM has greatly improved the proliferation potential of shrimp hemocytes.It was found that the proliferation percentages of shrimp hemocytes cultured in the optimal SGM were 30.61 ± 1.92% on the 10th day, but only 0.43 ± 0.14% on the 30th day by EdU staining [1] .

Table 4
The reagents required in EdU assay (not provided in the kit).Seen in Table 3 EdU (5-ethynyl-2 ′ -deoxyuridine) is a thymidine analogue which can be incorporated into the newly synthesized DNA.The acetylene group of EdU molecule can covalently react with fluorescence-labeled small molecular azide probe (such as Azide Alexa Fluor 488) through the catalysis of univalent copper ions and form a stable triazole ring, which is very rapid and thus called "click reaction ".Through the "click reaction ", the newly synthesized DNA will be labeled with the corresponding fluorescence probe, so that the proliferating cells can be detected using the appropriate fluorescence detection equipment [7] .The BeyoClick TM EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, China) was chosen for use in this study.The reagents required in the experiment but not provided in the kit were shown in Table 4 and the experimental steps were as follows in detail (take a 6-well plate as an example).
a.The medium containing 1 × EdU (10 μM), that is, EdU working solution, was added to a well which is seeded with MeH cells and incubated at 28 • C for 24 h.b.After EdU labeling, the culture medium was removed, and 1 mL of fixative was added and fixed for 15 min at room temperature.c.The fixative was removed and the cells were washed 3 times with 1 mL of washing solution per well, 3-5 min each time.d.The washing solution was removed, and each well was incubated with 1 mL permeabilization reagent for 10-15 min at room temperature.e.The permeabilization reagent was removed, and the cells were washed 1-2 times with 1 mL washing solution for 3-5 min each time.f.The click reaction solution needed to be ready-made.Added and mixed 430 μL click reaction buffer, 20 μL CuSO 4 , 1 μL azide 488 and 50 μL click additive solution in turn, and used the click reaction solution within 15 min.g.The washing solution from the step e was removed and then 0.5 mL of click reaction solution was added to each well, and the culture plate was gently shaken to ensure that the reaction mixture could cover the cells evenly.h.After incubation at room temperature for 30 min in the darkness, the click reaction solution was removed and the cells were washed with the washing solution for 3 times, 3-5 min for each time.i.In order to detect the cell proliferation percentage, Hoechst 33,342 was used for nuclear staining.1 × Hoechst 33,342 solution was prepared of by diluting the stock solution of Hoechst 33,342 (1000 ×) with PBS at the ratio of 1: 1000.j.After the step h, after the washing solution was removed, 1 mL of 1 ×Hoechst 33,342 solution was added to each well and incubated at room temperature in darkness for 15 min.k.The 1 ×Hoechst 33,342 solution was removed and the cells were washed with the washing solution for 3 times, 3-5 min each time.Then fluorescence detection can be carried out.
From the results of Calcein-AM staining and EdU staining in the related research article [1] , it is found that there is a drastic variation existed between cellular activity and proliferative activity of the MeH cells.It has been reported that shrimp cells usually stop dividing and enter a "quiescence state " once isolated and cultured in vitro within for 24 h, that is, the cells are alive but not mitotic [ 8 , 9 ].In the present study, the SGM has been found to be able to partially solve the problem of mitosis-arrest of shrimp hemocytes, but it is still unable to support the hemocytes to actively proliferate for too long time, which is the next important scientific question to be solved in the future.
There is no doubt that SGM can successfully supported the long-term in vitro culture of hemocytes of Metapenaeus ensis , but this medium has not been tested in other shrimp species.In consideration of the similar nutrient requirement, we believe that, this medium should be suitable for the hemocyte culture of other seawater penaeid shrimps and even freshwater penaeid shrimps after a minor modification of its osmolarity.For example, based on some literature reports, it was found that the content of proline, taurine and glucose in the sera of some seawater shrimps like Penaeus vannamei [10] and Penaeus stylirostris [11] was similar to that in the serum of Metapenaeus ensis .Of course, the applications of SGM medium in other shrimp species with more distant relationship like lobster and crayfish or other crustacean should be cautious and needs more experimental verification.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .
Fig. 1.Diagram of the preparation process of shrimp tissue extracts.

Fig. 2 .
Fig. 2. Diagram of the preparation process of the optimal shrimp cell growth medium.

Fig. 3 .
Fig. 3.The volume percentages of each of the stock solutions and other supplements in the optimal shrimp cell growth medium.

Fig. 4 .
Fig. 4. Diagram of the drawing of shrimp hemolymph using a 2.5-mL syringe.Panel A shows the sampling site at the ventral sinus between the pereiopods and pleopods of shrimps as indicated in red circle.Panel B shows the process of drawing out of shrimp hemolymph.

Table 2
The formulas of stock solutions used to prepare the optimal SGM.

Table 3
The formulas of anticoagulant solution (left) and 2 × PBS (right).